Lentiviral vectors have been used successfully to transduce various cell types, such as neurons, hepatocytes, haematopoietic stem cells, retinal cells, dendritic cells, myocytes and islet cells. Notable pre-clinical animal studies include successful correction of β-thalassaemia and sickle cell anaemia , haemophilia B and ZAP-70 (ζ -chain-associated protein kinase of 70 kDa) immunodeficiency , whereas amelioration of other major genetic disorders has been seen with lentiviral vector-mediated interventions, including Parkinson’s disease , cystic fibrosis and spinal muscular atrophy. In this section, we describe various lentiviral applications for basic and translational researches.
Delivery of complex genetic structures
Lentiviral vectors typically encode an internal promoter and a polyA-less cDNA in the sense orientation. However, owing to the Rev/RRE-mediated nuclear export of unspliced vector genome RNAs, they can efficiently transfer complex genetic structures by putting introns into the vector by placing the transgene expression cassette in backwards.
For generation of multigene lentiviral vectors, two strategies are typically employed to express multiple genes; through incorporation of multiple expression cassettes or construction of a polycistronic transcript driven by a single internal promoter. The use of multiple expression cassettes allows independent gene expression driven by different internal promoters. Some studies successfully used bi-directional promoters to express multiple genes. Owing to the limited packaging capacity of lentiviral vectors.
RNAi (RNA interference) is an evolutionarily-conserved gene silencing mechanism that is induced by dsRNA (doublestranded RNA). Since synthetic siRNAs or shRNAs (short hairpin RNAs) can suppress the expression of genes of interest in mammalian cells , RNAi-mediated gene silencing has become an essential technology to study gene functions. Importantly, lentiviral vectors can be engineered to achieve stable high-efficiency gene silencing in a wide variety of cells . In gene-silencing lentiviral vectors, siRNA can be delivered as a form of shRNA driven by a RNA polymerase III promoter or as a part of an miRNA-like structure expressed from a RNA polymerase II promoter.
Gene-silencing lentiviral vectors have been successfully used to inhibit HIV-1 infection, retard the onset and the progression rate of amyotrophic lateral sclerosis in mice , extend the survival of scrapie-developed mice and increase the expression of fetal haemoglobin. An important advance in the RNAi field is the completion of the generation of a silencing lentiviral library, which targets 15 000 human genes and 15 000 mouse genes, by the RNAi Consortium, a public–private consortrium based at the Broad Institute (http://www.broadinstitute.org/rnai/trc).
The genome-wide shRNA library vectors, which are based on a shRNA-carrying lentiviral vector pLKO.1, are available through Sigma–Aldrich and Open Biosystems. In pLKO.1 vector, an shRNA construct is expressed by a RNA polymerase III U6 promoter, whereas a separate puromycin-resistant gene-expression cassette allows selection of transduced cell populations. Similarly, pre-designed shRNA- or miRNA-carrying lentiviral vectors are also available from various vendors and are in rampant use in basic biology laboratories. The caveats of the use of gene-silencing lentiviral vectors are similar to other RNAi platforms; they can induce off-target gene silencing or undesired interferon response.
To rule out those possibilities, it is critical to perform RNAi rescue experiments involving expression of the target gene containing silent mutations in the RNAi-targeted sites. As an additional application of lentiviral vectors for studying gene silencing, Naldini and co-workers have developed lentiviral vectors overexpressing miRNA target sequences from polymerase II promoters and showed stable and specific knockdown of miRNA function in vivo.
Inducible vector system
Lentiviral vectors can be used for externally controllable transgene expression. Among several inducible systems, the rtTA (reverse tetracylin-controlled transactivator)-regulated system has been widely used for inducible gene expression . This system uses a chimaeric transcription factor tTA transactivator, a fusion protein between the bacterial TetR (Tet repressor) with the activating domain of herpes simplex virus VP16 (viral protein 16), or its derivative rtTA protein, along with a TetO-bindingsites-containing promoter for tetracycline-induced gene silencing (Tet-Off) or activation (Tet-On) respectively.
The Tet-On/Tet-Off systems have been studied extensively in the context of lentiviral vectors, in which the Tet-regulated system is incorporated into a single lentivector. Using a similar vector design, conditional gene-silencing lentiviral vectors have also been developed. To date, various inducible lentivector systems have been developed, and are commercially available. Since several studies have identified VP16, used in the Tet transactivator, as the key factor to induce non-specific gene induction or cytotoxicitiy, which can lead to data misinterpretation, a new doxycycline-regulated system based on the original TetR with no VP16 has been developed and incorporated into lentiviral vectors.
Lentiviral vector-mediated transgenesis
Lentiviral vectors can efficiently transduce embryonic stem cells. When compared with conventional retroviral vectors, lentiviral vectors are relatively resistant to gene silencing in mammalian embryonic stem cells. Accordingly, lentivirally modified murine embryonic stem cells have been used to generate transgenic mice. As an alternative approach, lentivectors have been used to introduce transgenes into early embryos. This technology has been highly successful and has led to the generation of various transgenic animals, including chicken, rat, cat and pig, in which reliable tissue-specific reporter gene expression has been observed after germline transmission. In addition, lentiviral vectors expressing siRNAs have also been used to knock down targeted genes in vivo.
Lentiviral vector-mediated immune modulation
Lentivectors can efficiently transduce antigen-presenting cells, including dendritic cells. This property has been exploited as lentiviral vaccines for tumours and infectious diseases. Lentivector-mediated expression of melanoma antigens or ovalubumin in dendritic cells elicits both CD8 + T-cells and CD4 + T-cell responses. Lentivector-induced tumour-specific immunity has demonstrated regression of tumours. For HIV/AIDS vaccine development, lentivector-mediated gene transfer has also induced Gag-specific T-cell responses.
Introduction of a set of defined pluripotency-associated factors, such as OCT4 (octamer 4), SOX2 (sex-determining region Ybox 2), KLF4 (Kruppel-like factor 4) and c-Myc (or OCT4, ¨ SOX2, NANOG and LIN28), in somatic cells has demonstrated the generation of embryonic stem cell-like pluripotent stem cells. Since multiple factors have to be introduced into a single cell, multiple lentiviral vectors have been used for successful reprogramming of various cell types, such as fibroblasts, keratinocytes and haematopoietic stem cells.
To increase the reprogramming efficiency and safety, single polycistronic lentiviral vectors carrying the four reprogramming factors linked by self-cleaving 2A peptides have been developed, some of which with self-deleting properties. Owing to the concerns of reactivation of pluripotency-associated factors in iPS (induced pluripotent stem cell) progeny, the field has been shifted towards other reprogramming technologies which do not require integrating vectors.
In addition to the pluripotency induction, lentiviral vectors have been used to trans-differentiate adult somatic cells into other types of cells. For instance, lentiviral introduction of Gata4, Hnf1a and Foxa3 (Forkhead box a3) in murine fibroblasts results in generation of hepatocyte-like iHep cells, whereas ectopic expression of Gata4, Mef2c and Tbx5 in murine fibroblast cells can trans-differentiate fibroblasts into functional cardiomyocytes.
In vivo imaging and lineage tracking
Lentiviral vector-mediated stable gene transduction can be exploited for in vivo monitoring/live imaging of vector-infected cells . Cancer cells, which are engineered to express reporter genes by lentiviral vectors, can be monitored for their growth or metastasis in vivo, whereas transplantation of lentivirally modified bone marrow cells/stem cells allows the identification of stem cell-derived progeny in vivo. Moreover, stem cell transduction by a vector with a tissue-specific promoter allows lineage tracking in vitro and in vivo.
For instance, lentiviral vectors with a marker gene under the control of a troponin-I promoter can be used to track cardiomyocyte differentiation of human embryonic stem cells. Recently, Gaussia luciferase fragment complentation has been incorporated into lentiviral vectors for monitoring ligand–receptor binding in vivo.
Lentiviral vectors in the clinic
The first Phase I clinical trial using a HIV-based lentiviral vector expressing an antisense gene against HIV env was started for AIDS gene therapy in 2003. In that study, a VSV-G-pseudotyped vector with no HIV-1 accessory proteins, named VRX496, was produced from an advanced two-plasmid packaging system. Five subjects who received a single dose (∼1010 cells) of VRX496-transduced autologous CD4 + Tcells resulted in an increase of CD4 + T-cells (four out of five subjects) and decrease of viral load (five out of five participants) after 1 year.
Lentiviral vector-mediated gene therapy for haematopoietic stem cells has also demonstrated initial clinical benefits in X-linked adrenoleukodystrophy. A Phase I/II clinical trial of β-globin gene therapy for β-thalassaemia also began in 2007. A self-inactivating lentivector, LentiGlobin, contains large elements of the β-globin locus control region, and chromatin insulators (cHS4) were used to transduce CD34 + cells. After approximately 3 years of treatment, the patients have corrected β-globin gene and stable blood haemoglobin levels. No insertional mutagenesis has been reported from both trials so far, and these studies provide therapeutic benefit for the patients.
As summarized above, considerable efforts have been made to increase the safety and efficacy of lentiviral vector systems. Currently available vectors are mostly generated from multiple plasmids, with no HIV-1 accessory gene and with self-inactivating modification, yet show high gene transduction efficiency in vitro and in vivo. It is also notable that no RCL has been observed in third-generation HIV-1 vectors. Because of their increased safety and proven efficiency, we can now purchase ready-touse lentivector particles from various vendors. It is likely that lentivector technology will evolve further and expand into new research fields where the vector users may have a limited background in lentiviruses. This state-of-the-art system represents a huge success for lentiviruses as a gene transfer vector system.
However, it should be noted that these vectors are derived from pathogenic viruses. The possibility of generating an RCL may be very low, but may not be zero, where a single infection event of an oncogene-carrying lentiviral vector may be enough to induce tumours. When sufficient precautions are taken, the lentiviral vector technology will provide an effective and versatile platform which achieves various creative and innovative applications.